Rational design of antibodies and development of a novel method for (1-3)-ß-D glucan detection as an alternative to Limulus amebocyte lysate assay.

With advances in medicine, increasing medical interventions have increased the risk of invasive fungal disease development. (1-3)-ß-D glucan (BDG) is a common fungal biomarker in serological tests. However, the scarcity of Limulus resources for BDG detection poses a challenge. This study addresses the need for an alternative to Limulus amebocyte lysate by using BDG mutant antibody for chemiluminescence detection. The wild-type BDG antibody was obtained by immunizing rabbits. An optimal V52HI/N34L Y mutant antibody, which has increased 3.7-fold of the testing efficiency compared to the wild-type antibody, was first achieved by mutating "hot-spot" residues that contribute to strong non-covalent bonds, as determined by alanine scanning and molecular dynamics simulation. The mutant was then applied to develop the magnetic particle chemiluminescence method. 574 clinical samples were tested using the developed method, with a cutoff value of 95 pg/mL set by Limulus amebocyte lysate. The receiver operating characteristic curve demonstrated an area under the curve of 0.905 (95% CI: 0.880-0.929). Chemiluminescence detected an antigen concentration of 89.98 pg/mL, exhibiting a sensitivity of 83.33% and specificity of 89.76%. In conclusion, the results showed a good agreement with Limulus amebocyte lysate and demonstrated the feasibility of using BDG mutant antibodies for invasive fungal disease diagnosis. The new method based on chemiluminescence for detecting BDG could shorten the sample-to-result time to approximately 30 min, rescue Limulus from being endangered and is resource efficient in terms of equipment and the non-use of a skilled technician.

Time-resolved fluoroimmunoassay for Aspergillus detection based on anti-galactomannan monoclonal antibody from stable cell line.

Invasive Aspergillosis is a high-risk illness with a high death rate in immunocompromised people due to a lack of early detection and timely treatment. Based on immunology study, we achieved an efficient production of anti-galactomannan antibody by Chinese hamster ovary (CHO) cells and applied it to time-resolved fluoroimmunoassay for Aspergillus galactomannan detection. We first introduced dual promoter expression vector into CHO host cells, and then applied a two-step screening strategy to screen the stable cell line by methionine sulfoximine pressurization. After amplification and fermentation, antibody yield reached 4500 mg/L. Then we conjugated the antibodies with fluorescent microspheres to establish a double antibody sandwich time-resolved fluoroimmunoassay, which was compared with the commercial Platelia™ Aspergillus Ag by clinical serum samples. The preformed assay could obtain the results in less than 25 min, with a limit of detection for galactomannan of approximately 1 ng/mL. Clinical results of the two methods showed that the overall percent agreement was 97.7% (95% CI: 96.6%-98.4%) and Cohen's kappa coefficient was 0.94. Overall, the assay is highly consistent with commercial detection, providing a more sensitive and effective method for the rapid diagnosis of invasive aspergillosis.

Development and evaluation of RPA-NFO-LFT and RPA-Cas12a-LFT systems for the detection of Candida albicans.

Recently, the growing number of medical interventions has led to the risk of invasive candidiasis. Among them, Candida albicans (C. albicans) infection has the highest incidence, which has led to great demand for developing early diagnosis methods. In this study, two lateral flow device based molecular assay systems, RPA-NFO-LFT and RPA-Cas12a-LFT, were established and optimized to achieve the detection of C. albicans. Firstly, efficient and specific primers for C. albicans detection were designed and screened, and the purification of amplification products was also explored. Then, many important conditions and issues for each system were investigated and discussed to improve the performances of the test strip devices in C. albicans detection. An evaluation study revealed that both systems showed favorable specificity and sensitivity in the detection of C. albicans samples with a lower detection limit of 103 CFU ml-1, while RPA-Cas12a-LFT is more accurate for visual interpretation and more stable toward samples that exhibit serum nucleic acid interference. Finally, the performances of RPA-NFO-LFT and RPA-Cas12a-LFT were compared with that of the conventional qPCR method. This work might provide a reference for the development of molecular assay devices in practical candidiasis diagnosis.

High Production of γ-Aminobutyric Acid by Activating the xyl Operon of Lactobacillus brevis.

γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter with important physiological functions such as sleep assistance and anti-depression. In this study, we developed a fermentation process for the high-efficiency production of GABA by Lactobacillus brevis (Lb. brevis) CE701. First, xylose was found as the optimal carbon source that could improve the GABA production and OD600 in shake flasks to 40.35 g/L and 8.64, respectively, which were 1.78-fold and 1.67-fold of the glucose. Subsequently, the analysis of the carbon source metabolic pathway indicated that xylose activated the expression of the xyl operon, and xylose metabolism produced more ATP and organic acids than glucose, which significantly promoted the growth and GABA production of Lb. brevis CE701. Then, an efficient GABA fermentation process was developed by optimizing the medium components using response surface methodology. Finally, the production of GABA reached 176.04 g/L in a 5 L fermenter, which was 336% higher than that in a shake flask. This work enables the efficient synthesis of GABA using xylose, which will provide guidance for the industrial production of GABA.

Rational design of tryptophan hydroxylation 1 for improving 5-Hydroxytryptophan production.

5-Hydroxytryptophan (5-HTP) is a chemical precursor of serotonin, which synthesizes melatonin and serotonin in animals and regulates mood, sleep, and behavior. Tryptophan hydroxylase (TPH) uses tetrahydrobiopterin (BH4) as a cofactor to hydroxylate L-tryptophan (L-Trp) to 5-HTP, and the low catalytic activity of TPH limits the rate of hydroxylation of L-Trp. In this study, the catalytic mechanism and structural features of L-Trp-TPH1-BH4 were investigated, and the catalytic activity was improved using a rational design strategy. Then the S337A/F318Y beneficial mutation was obtained. Molecular dynamics simulations showed that the S337A/F318Y mutant formed a salt bridge with TPH1 while forming an additional hydrogen bond with the substrate indole ring, stabilizing the indole ring and enhancing the binding affinity of the variant to L-Trp. As a result, the yield of 5-HTP was increased by 2.06-fold, resulting in the production of 0.91 g/L of 5-HTP. The rational design of the TPH structure to improve the hydroxylation efficiency of L-Trp offers the prospect of green production of 5-HTP.

The safe Laccase@ZIF-8-prodrug system with GSH redox cycle for effective targeted cancer therapy with low off-target toxicity.

Nanotechnology has been widely used in cancer treatment but only a small fraction (0.7 %) of the administered nanoparticle was delivered into a solid tumor, while the remaining fraction causes off-target toxicity in healthy tissues. The activation of prodrugs by exogenous enzymes can be used as an effective anticancer strategy to reduce systemic toxicity. In this study, the laccase@zeolitic imidazolate framework-8 (LA@ZIF-8) enzyme-activated prodrug system was created based on the high catalytic activity of LA in an acidic environment and the pH response (pH∼5.5) of ZIF-8. Quercetin (QU) was selected as the prodrug, which is non-toxic and even beneficial without being activated by LA. LA could be precisely released in the acidic tumor microenvironment for activating nontoxic QU successfully to produce toxic oxidized quercetin (OQU), and the LA was steady loaded on the LA@ZIF-8 under physiological conditions (pH∼7.4). Especially, the high concentrations of glutathione (GSH) in tumors could accelerate the oxidation of QU by LA. Meanwhile, GSH could be consumed continuously by the reduction of OQU for regenerating QU, thus a LA-QU-GSH redox was formed ingeniously. Therefore, the synergistic effect of OQU toxicity and GSH depletion induced reactive oxygen species (ROS) accumulation and tumor cell apoptosis. Overall, the LA@ZIF-8-QU prodrug system provides ideas for safe and effective cancer treatment with low off-target toxicity.

Study of the kinetics and mechanism of ferrocene-tripeptide inhibiting insulin aggregation.

Peptides are gaining popularity as neurodegenerative disease-targeting drugs due to their medicinal value and simplicity in the biomedical and pharmaceutical industry fields. Herein, based on previously studied ferrocene-modified dipeptide (Fc-dipeptide) ferrocene-L-Phe-L-Phe (Fc-FF), we developed four Fc-tripeptides, i.e., Fc-FFY, Fc-FFF, Fc-FFD, and Fc-FFK, to further study the anti-amyloid effects of peptide-based inhibitors. The results showed that all Fc-tripeptides inhibited the formation of insulin fibrils in a dose-dependent manner more responsive than Fc-FF. Meanwhile, Fc-FFY and Fc-FFF had a more significant inhibitory effect on insulin amyloid fibrillation than Fc-FFD and Fc-FFK. In addition, molecular dynamics simulations indicated that Fc-FFY and Fc-FFF were contacted mainly with the hydrophobic core residues of insulin chain A and chain B, respectively. The research indicated that Fc-tripeptides have potential effects in preventing protein misfolding diseases and could be further used to design effective anti-amyloidosis compounds.

Enhancement of the polyethylene terephthalate and mono-(2-hydroxyethyl) terephthalate degradation activity of Ideonella sakaiensis PETase by an electrostatic interaction-based strategy.

The serious environmental pollution that came up with the continuously growing demand for polyethylene terephthalate (PET) has attracted global concern. The IsPETase which has shown the highest PET degradation activity under ambient temperature is a promising enzyme for PET biodegradation, while poor thermostability limited its practical application. Herein, an electrostatic interaction-based strategy was applied for rational design of IsPETase towards enhanced thermostability. The IsPETaseI139R variant displayed the highest Tm value of 56.4 °C and 3.6-times higher PET degradation activity. Molecular simulations demonstrated that the introduction of salt bridges stabilized the local structures, resulting in robust thermostability. Meanwhile, the IsPETaseS92K/D157E/R251A not only exhibited higher thermostability but also showed a 1.74-fold kcat increase towards mono-(2-hydroxyethyl) terephthalate, which ultimately achieved PET depolymerization to complete monomer TPA. Collectively, the electrostatic interaction-based strategy, together with the derived IsPETase variants, could help promote the bio-recycle of PET, reducing the severe global burden of PET waste.

Copper ions binding regulation for the high-efficiency biodegradation of ciprofloxacin and tetracycline-HCl by low-cost permeabilized-cells.

Cu2+ plays a decisive role for the bio-oxidation in the active center of laccase. In the fermentation-purified process, the loss of Cu2+ reduces the activity and the high cost limits the application of laccase. In this study, a fermentation-permeabilization combined process were developed which based on the regulation of Cu2+ binding time to produce the permeabilized-cells containing laccase, in which Cu2+ can enter the cells freely to greatly improve the laccase activity and reduce the immobilization cost by about 19 times. So, the permeabilized-cells is suitable for biodegradation of antibiotic pollution in the environment, which was applied for the biodegradation of ciprofloxacin (CIP) and tetracycline-HCl (TCH) and the degradation efficiency reached 95.42% and 98.73%, respectively, with low ecotoxicity of the degradation products. Finally, the degradation mechanism was analyzed theoretically by molecular docking. Therefore, this study provided a low-cost, eco-friendly, and widely applicable method for organic pollutants removal.

Laccase-catalyzed soy protein and gallic acid complexation: Effects on conformational structures and antioxidant activity.

The ability of laccase to oxidize polyphenols arouses our interest that laccase can be applied for protein-polyphenol cross-linking. In this study, laccase promoted the cross-linking of gallic acid (GA) and soy protein isolate (SPI) under neutral pH. SPI-GA complexes changed the secondary structures with a decrease in ß-fold and an increase in α-helix and ß-turn. The free-radical scavenging activity and reducing power determination results suggested that GA elevated the SPI antioxidant activity significantly. Specifically, DPPH free radical scavenging rate and ABTS free radical scavenging ability increased almost 5- and 1.5-fold compared with unmodified SPI, respectively. Moreover, the reducing power had more than 3-fold compared to the SPI control. This study provided a novel enzyme-induced approach to modulate the physicochemical properties of SPI binding polyphenol.

Rational design of 17ß-hydroxysteroid dehydrogenase type3 for improving testosterone production with an engineered Pichia pastoris.

Testosterone (TS) is a critical androgenic steroid that regulates human metabolism and maintains secondary sexual characteristics. The biotransformation from 4-androstene-3,17-done (4-AD) to TS is limited by the poor catalytic activity of 17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3). Herein, we explored the structural characteristics and catalytic mechanism of 17ß-HSD3 and adopted the rational design strategy to improve its catalytic activity. Molecular docking and molecular dynamics simulations revealed the substrate-binding pocket and the binding mode of 4-AD to 17ß-HSD3. We located the pivotal residues and regulated their hydrophobicity and polarity. The obtained G186R/Y195W variant formed additional electrostatic interaction and hydrogen bond with 4-AD, increasing the binding affinity between the variant and 4-AD. Therefore, the G186R/Y195W variant produced 3.98 g/L of TS, which increased to 297%. The combination of structural and mechanism resolution drives the implementation of the rational design strategy, which provides guidance for bioproduction of TS catalyzed by 17ß-HSD3.

Bifunctional utilization of whey powder as a substrate and inducer for ß-farnesene production in an engineered Escherichia coli.

ß-Farnesene can replace petroleum products as specialty fuel to solve the global fuel energy crisis, but its production by Escherichia coli (E.coli) using glucose and isopropyl ß-D-1-thiogalactopyranoside (IPTG) is costly. Hence, we developed a new strategy to produce ß-farnesene by engineered E.coli strain F13 with bifunctional utilization of whey powder. The utilization of whey powder as a substrate ensured the growth of the strain F13, while whey powder could also replace IPTG to induce the production of ß-farnesene. In shake flasks, ß-farnesene production reached 2.41 g/L by the bifunctional utilization of whey powder as a substrate and inducer, 65.1% higher than that with IPTG and glucose. In the 7 L bioreactor, ß-farnesene production reached 4.74 g/L using whey powder, which was 197% of that in shake flasks. Therefore, this new strategy might be an attractive route to broaden the applications of whey powder and achieve the economical production of ß-farnesene.

Self-Assembly of Peptide Hierarchical Helical Arrays with Sequence-Encoded Circularly Polarized Luminescence.

Self-assembled peptide materials with sequence-encoded properties have attracted great interest. Despite their intrinsic chirality, the generation of circularly polarized luminescence (CPL) based on the self-assembly of simple peptides has been rarely reported. Here, we report the self-assembly of peptides into hierarchical helical arrays (HHAs) with controlled supramolecular handedness. The HHAs can emit full-color CPL signals after the incorporation of various achiral fluorescent molecules, and the glum value is 40 times higher than that of the CPL signal from the solutions. By simply changing the amino acid sequence of the peptides, CPL signals with opposite handedness can be generated within the HHAs. The peptide HHAs can provide hydrophobic pockets to accommodate the fluorescent molecules with helical arrangement through strong aromatic stacking interactions, which are responsible for the CPL signals. This work provides a pathway to construct highly ordered chiral materials, which have broad applications in the chiroptical field.

Self-Templated, Enantioselective Assembly of an Amyloid-like Dipeptide into Multifunctional Hierarchical Helical Arrays.

Chiral self-assembly of peptides has attracted great interest owing to their promising applications in biomedicine, chemistry, and materials science. However, compared with the rich knowledge about their chiral self-assembly at the molecular or nanoscale, the formation of long-range-ordered hierarchical helical arrays (HHAs) from simple peptides remains a formidable challenge. Herein, we report the self-templated assembly of an amyloid-like dipeptide into long-range-ordered HHAs by their spontaneous fibrillization and hierarchical helical assembly within a confined film. The chiral interactions between the peptide and diamines result in geometry frustration and the phase transition of self-assembling peptide films from achiral spherulite structures into chiral HHAs. By changing the chirality and enantioselective interactions, we can control the phase behavior, handedness, and chiroptics of the self-assembled HHAs precisely. Moreover, the redox activity of the HHAs allows the in situ decoration of nanoparticles with high catalytic activity. These results provide insights into the chiral self-assembly of peptides and the fabrication of highly ordered materials with complex architectures and promising applications in chiroptics and catalysis.

An effective enzymatic assay for pH selectively measuring direct and total bilirubin concentration by using of CotA.

In this study, we aimed to develop B. subtilis spore coat protein A (CotA) for the enzymatic determination of bilirubin. Firstly, molecular docking and oxidation kinetic analysis confirmed the feasibility of CotA for oxidizing bilirubin. Secondly, CotA showed pH-preferable oxidization performance to direct bilirubin (DB) in acidic conditions and an alkaline-catalytic oxidation capacity to total bilirubin (TB). Mechanism analysis results confirm that the conformational changes of CotA, DB and UB caused by pH changes are responsible for the selective oxidation of DB and TB by CotA. Then, CotA exhibits better structural characteristics and enzymatic performance than M. verrucaria-derived bilirubin oxidase (Mv-BOD). Besides, the strong anti-interference ability helps CotA adapt to complex catalytic environment in the detection of DB and TB. Our results prove that CotA can be used as a promising candidate bio-enzymatic detection reagent for DB and TB.

Investigation of fermentation conditions of biodiesel by-products for high production of ß-farnesene by an engineered Escherichia coli.

Recently, the research on conversion of biodiesel by-products to high value-added products has received much attention, due to the adverse effects of large accumulations of biodiesel by-products caused by the rapid increase in biodiesel production. Herein, this study investigated the utilization of by-products crude glycerol (CG-1 and CG-2) from two different industrial methods of biodiesel production and the favorable fermentation conditions for the high yield of ß-farnesene by an engineered Escherichia coli F4, which harbored an optimized mevalonate pathway. Through analyzing by-products' components and fermentation performance, we found that CG-2 did not contain harmful impurities such as methanol and black solid impurities, and the ß-farnesene production was up to 2.7 g/L from CG-2, which was similar to that from pure glycerol (2.5 g/L) and higher than that (2.21 g/L) from CG-1. Therefore, CG-2 was more suitable for ß-farnesene production than CG-1, which might provide a reference for choosing a more suitable method on practical biodiesel production. Afterward, a variety of important fermentation conditions were explored using CG-2 as a substrate in shaken flasks. Under the optimal conditions (including induced cell density 1.0, initial cell density 0.25, temperature after induction 33 °C, initial medium pH 6.5), the yield of ß-farnesene from CG-2 reached 10.31 g/L in a 5-L bioreactor, which was 2.8-fold higher than initial conditions in shake flasks and was the highest yield of ß-farnesene produced from biodiesel by-products by fermentation as well. The recommended fermentation conditions in this work will provide a valuable reference for the industrial production of ß-farnesene utilizing biodiesel by-products.

An effective in-situ method for laccase immobilization: Excellent activity, effective antibiotic removal rate and low potential ecological risk for degradation products.

In this study, we used a simple in-situ biomineralization method to immobilize Bacillus subtilis (B. subtilis)-derived laccase into the copper-Trimesic acid framework (Cu-BTC), and the synthesized Laccase@Cu-BTC particles were used to degrade tetracycline and ampicillin. Compared with free laccase, the Laccase@Cu-BTC showed 16.5-fold of activity recovery, higher thermo-tolerant performance, more excellent acid-proof ability and reusability. Without any mediators, Laccase@Cu-BTC displayed high degradation efficiency (nearly 100%) for tetracycline and ampicillin in some actual water. The degradation mechanism and proposed degradation pathways of tetracycline and ampicillin were discussed technically. Besides, bacteriostatic assay and survival test of Escherichia coli (E. coli) and B. subtilis confirmed the loss of antibiotic activity for tetracycline and ampicillin, as well as the low ecotoxicity of the degradation products. Our research demonstrates that Laccase@Cu-BTC has excellent performance in the effective removal of antibiotics and the detoxification of degradation products, which make it a promising candidate for environmental recovery.

Ferrocene-modified peptides as inhibitors against insulin amyloid aggregation based on molecular simulation.

Peptide-based inhibitors have gradually been implicated as drugs for treating protein folding diseases because of their favorable biocompatibility and low toxicity. To develop potential therapeutic strategies for amyloid-related disorders, short peptides modified by Fc, ferrocene-l-Phe-l-Phe (Fc-FF) and ferrocene-l-Phe-l-Tyr (Fc-FY), were used as inhibitors for the investigation of the aggregation behavior of insulin. Firstly, molecular docking predicted the interaction between both Fc-peptides and insulin. Then, the experimental data from ThT, DLS, CD and TEM confirmed that Fc-FF and Fc-FY effectively inhibited insulin fibrillation and disaggregated mature insulin fibrils. Based on a dose-dependent manner, both Fc-peptides can strongly inhibit insulin fibrillation, extend lag phase time, reduce final fibril formation (beyond 99% by Fc-peptides of 400 µM), decrease the formation of high-content ß-sheet structures and reduce the size of insulin fibrils. Additionally, we found that compared with Fc-FY, the better inhibitory effect of Fc-FF at concentration below 400 µM was mainly resulted from the difference in π-π interaction and hydrogen bonds between Fc-peptides and insulin, according to molecular dynamics analysis. Our results demonstrated Fc-peptides, Fc-FF and Fc-FY, may play effective roles in the development of new therapeutic drugs or strategies for amyloid-related disorders, and the molecular dynamics simulation may be helpful for designing appropriate inhibitors of anti-amyloidosis diseases.

Construction of luffa sponge-based magnetic carbon nanocarriers for laccase immobilization and its application in the removal of bisphenol A.

The raw material of resin, Bisphenol A (BPA), is an endocrine-disrupting compound that can be continuously released into the environment and directly harms health. In this study, luffa sponge was used as the raw material to prepare magnetic carbon chemicals for laccase immobilization and BPA degradation. The MLC-1 was synthesized by one-step carbonization-magnetization method, which showed good magnetic properties and a strong load capacity for laccase. Compared with free laccase, Laccase@MLC-1 showed stronger thermal stability, better acid-tolerate performance and reusability. Moreover, Laccase@MLC-1 showed higher BPA degradation efficiency than free laccase. 100 mg/L of BPA can be completely removed by Laccase@MLC-1 in 4 h, while only 62.70% of BPA was removed by the same amount of free laccase. By improving reuse strategies, a complete BPA degradation ratio was obtained in each reoperation process. All results proved that Laccase@MLC-1 might be a suitable biocatalyst candidate for BPA removal.

High-efficiency and low-cost production of cadaverine from a permeabilized-cell bioconversion by a Lysine-induced engineered Escherichia coli.

Cadaverine is the monomer of bio-based nylons polyamide 5.4, 5.6 and 5.10. In this study, a litre-scale integrated strategy was developed for high-efficiency and low-cost production of cadaverine using an engineered Escherichia coli. Firstly, the engineered strain BL21-Pcad-CadA induced by cheap l-lysine-HCl instead of IPTG was constructed. Then the permeabilized cells were served as the biocatalyst for the production of cadaverine, because the enhanced permeability facilitated the mass transfer of the substrate and the release of products. After the replacement of industrial materials and the solution of the scale-up permeabilization process, cadaverine concentration reached 205 g/L with the yield of 92.1% after 20 h in a 2 L bioconversion system, achieving the level of industrial production. Furthermore, the costs of industrial materials for 2 L integrated strategy ($2.78) was only 1/11 of the lab reagents ($30.88). Therefore, the proposed strategy is a promising candidate for the industrial process of cadaverine.